Intense light unleashes male–male courtship behaviour in wild-type Drosophila

Drosophila courtship studies have elucidated several principles of the neurogenetic organization of complex behaviour. Through an integration across sensory modalities, males perform stereotypic patterns of chasing, courtship song production and copulation attempts. Here we report a serendipitous finding that intense light not only enhances courtship toward female targets but also triggers unexpected courtship behaviours among male flies. Strikingly, in wild-type male-only chambers, we observed extreme behavioural manifestations, such as 'chaining' and 'wheeling', resembling previously reported male-male courtship behaviours in fruitless mutants and in transformants with ectopic mini-white+ overexpression. This male-male courtship was greatly diminished in a variety of visual system mutants, including disrupted phototransduction (norpA), eliminated eye-colour screening pigments (white), or deletion of the R7 photoreceptor cells (sevenless). However, light-induced courtship was unhampered in wing-cut flies, despite their inability to produce courtship song, a major acoustic signal during courtship. Unexpectedly the olfactory mutants orco and sbl displayed unrestrained male-male courtship. Particularly, orco males attained maximum courtship scores under either dim or intense light conditions. Together, our observations support the notion that the innate male courtship behaviour is restrained by olfactory cues under normal conditions but can be unleashed by strong visual stimulation in Drosophila.

Here, we describe a serendipitous finding of male-male courtship behaviours in wild-type (WT) Drosophila melanogaster evoked by intense light. We observed the light intensitydependent increase in chasing, wing extension, courtship song, and chaining in different strains of WT males, using LED, incandescent, and sunlight illumination. We examined the phenomena in flies with genetic and surgical manipulations to elucidate the key roles of the visual and olfactory systems in gating this male-male courtship behaviour.

Intense light triggers courtship behaviour in male flies
A modified semi-automated Drosophila tracking system, IowaFLI Tracker [40], was employed to analyse activity in each circular arena containing eight flies (figure 1). The tracking system consisted of a clear polyacrylic sheet containing four arenas which were housed in a light-shielded cylindrical chamber equipped with an inner circular strip of LED lighting (figure 1a). A webcam mounted on top of the ceiling captured fly behaviours. In male-only arenas, we unexpectedly discovered a high frequency of courtship behaviours at intense light setting. Seconds after light on, we regularly observed chasing and attempting to court among male flies. Strikingly, we often saw chains of courting males (figure 1b; electronic supplementary material, movie S1), reminiscent of phenotypes previously found in mini-white transgenic lines [38,39] and fruitless mutants [25,41]. To quantify light-induced courtship, we developed a protocol where the eight flies in the arena were first subjected to 2-min illumination of relatively low intensity (0.4 klx, within the normal range of room lighting) followed by 2 min of highintensity light (18 klx) and subsequently, another 2-min lowintensity light period (figure 1d). For each successive 10-s interval, we manually scored the occurrence (0 or 1) of chasing, wing extensions and chaining in the arena. The total number of intervals during which the respective behaviours were  figure S1). (d) Quantification of light-induced male-male courtship behaviours. Eight males were subjected to a 2-min low-light intensity period, followed by a 2-min high-intensity light, and a 2-min low-light recovery period. For each successive time bin (10 s), the presence or absence of respective behaviours was scored (1 or 0). Data points presented show the average score pooling from 12 ROIs for the various behaviours (see Methods).
Error bars indicate SEM. (e) Total scores in (d) are summed over 2-min periods and shown with SEM and ROIs. One-way ANOVA, Bonferroni-corrected t-test post hoc was applied for comparisons between first low-light and high-light, as well as between high-light and the second low-light. *p < 0.05, **p < 0.01, ***p < 0.001.
observed in the arena was reported as the 'total score' (ranging from 0 to 12 for the 2-min period, figure 1d,e; see Methods). During the high-light period, the increase in total chasing score was more than 700%, approximately 400% for wing extension, and greater than 800% for chasing (figure 1e). In a modified arena equipped with a microphone, we confirmed the light-induced wing extension behaviour was related to song production among male flies (figure 1c; electronic supplementary material, figure S1). We found that like malefemale courtship songs, male-male songs consisted of pulse and sine songs. The frequency and duration of sine songs as well as the inter-pulse intervals and number of pulses per bout of the pulse song were analysed, showing overlapping ranges of these parameters between male-male and male-female songs (electronic supplementary material, figure S1b).

Generality and specificity of light-induced courtship behaviours
To determine whether light-induced male-male courtship is a specific phenomenon restricted to our WT Canton-S (CS) strain used in most of the experiments, we tested another WT strain of melanogaster, Berlin, and found the same male-male courtship behaviours as shown in figure 2a. We observed similar high-light-induced increases in wing extension and chasing behaviour as in CS flies, with occasional chaining observed as well (figure 2a). We also observed mixed-sex arenas (4 male and 4 female flies) to examine how high-intensity illumination affects malefemale interactions, to determine whether the intense light-triggered courtship behaviour was restricted towards other males only. As shown in figure 2b, in these mixedsex arenas, high-intensity lighting also similarly increased male-female courtship behaviours (approx. 300%). In a subset of mixed-sex arenas, we determined the kinetics of intense-light effect on male-female courtship scores and found temporal properties similar to male-only arenas (electronic supplementary material, figure S2). Together, these findings suggest intense light intensifies courtship drive of male flies towards both male and female flies, rather than switches their sex preference from females to males. . Note the light-induced increase in courtship behaviour is absent in visual mutants, but unimpaired in flies with one (1x) or both (2x) wings removed. Olfactory mutants show greatly enhanced courtship activity, with orco attaining nearly maximum scores even at low light intensity. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, paired t-test (high versus low). For each panel, the sample size (ROIs) is indicated.
royalsocietypublishing.org/journal/rsob Open Biol. 13: 220233 2.3. Roles of sensory cues in light-triggered male-male courtship We characterized the efficacy of different lighting conditions for triggering the male-male phenomenon. By adjusting the arena LED light intensity, we found that the relationship between light intensity and incidence of wing extensions, chasing and chaining behaviours was monotonic and that the proportionally steepest increases occurred between 2.0 and 6.0 klx (electronic supplementary material, figure S3). We then asked whether the LED light source we used was specifically required, or some other sources could also evoke the effect. As shown in figure 2c, intense incandescent light (25 klx) also elicited courtship behaviour. We further asked if natural light (e.g. sunlight on a spring day) could induce male-male courtship activity. Remarkably, after moving the arena from a shaded location (approx. 0.4 klx) to a location under direct bright sunlight (approx. 90 klx), we observed robust courtship activity (figure 2c). These results indicate that a wide variety of sufficiently intense, broad-spectrum visible light sources (see electronic supplementary material, figure S4, for spectral measurements of different light sources) can effectively induce male-male courtship behaviour. We introduced experimental perturbations to the various sensory systems for indications of their roles in the lightinduced male-male courtship. First, we examined males with a disrupted visual system. The gene norpA encodes the enzyme phospholipase C required for phototransduction, and several mutations eliminate photoreceptor potential, rendering mutant flies completely blind [42]. In blind norpA P12 mutants, we found intense light did not induce courtship activity (figure 2d; electronic supplementary material, movie S1).
We further took advantage of anatomical mutations affecting a particular category of photoreceptors. The Drosophila compound eye is composed of about 800 facets, each cylindrical rhabdomere below consisting of 6 outer photoreceptors (R 1 -R 6 , green light sensitive) and two central photoreceptors (R 7 and R 8 , UV and blue light, respectively). In sevenless (sev) mutants, the high-acuity channels composed of central R 7 and R 8 are functionally impaired because R 7 is absent, which also disrupts the light guide for R 8 underneath, while the high sensitivity channel, consisting of R 1 -R 6 , is intact [43]. The results showed that light-evoked male-male courtship was absent in sev mutants (figure 2d), implying a critical role for the R 7 /R 8 system in light-triggered malemale courtship. Visual processing depends on integration of photoreceptor responses from individual rhabdomeres. Because of the screening pigments, each rhabdomere acts as an independent light guide for input from a narrow angular visual field, in isolation of its neighbours [44]. Mutations of the white (w) gene are devoid of the screening pigments and thus degrade visual image processing, in spite of increased sensitivity to light [45]. In w mutant alleles, including w 1118 and w G , despite their active locomotion in the arena, the male-male courtship behaviour was missing (electronic supplementary material, figure S5). Conceivably, visual acuity is crucial to the locomotion control during courtship. It is documented that white mutant males show lower performance in male-female courtship [46,47]. Further, additional eye colour mutants have been reported to be defective in courtship behaviour [48]. We have examined cinnabar (cn) and cinnabar brown (cn bw). The cn flies lack the screening pigment ommochrome [49] and show bright orange eye colour; bw lack pteridine [49] and cn bw flies are white-eyed. Our initial observations suggest that both cn and cn bw mutants showed greatly decreased intense-lightinduced male-male courtship, comparable to the w alleles described above. These observations indicate the central role of visual function in the light-evoked male-male courtship.
By contrast to the visual system, manipulations of mechanosensory or auditory cues revealed drastically different outcomes. In groups of males, courtship behaviours are known to be triggered by playback of courtship song [19]. However, we found that in wing-cut male flies, which had disrupted wing mechanosensory inputs and were unable to produce courtship song, intense light nevertheless reliably triggered chasing and chaining (figure 2e). Notably, flies with one wing or both wings cut displayed similar chasing and chaining scores and flies with only a single wing could attain a frequency of wing extension comparable to WT. This result demonstrates that the auditory cues generated by wing beats and the mechanosensory receptors along the wing blades are not required in the light-evoked male-male courtship.
To determine the role of olfactory systems, we examined two mutants: smellblind (sbl), a para Na + channel allele with disrupted odour-evoked behaviours [50], and odorant receptor co-receptor (orco), encoding a requisite olfactory receptor subunit [51]. Surprisingly, at low light intensities, both mutants displayed strikingly elevated courtship behaviours compared to WT males (figure 2f ). Particularly, recurrent male-male courtship in orco flies was so prevalent (electronic supplementary material, movie S1), frequently approaching the maximum score of 12, that a further increase in courtship was not apparent during the intense light periods. However, in sbl males, which showed milder increases in low-light scores, intense light could further promote courtship activities to attain the maximum score (figure 2f ), prompting a further investigation into interactions between intense light and olfactory processing in gating male courtship behaviours.
Apparently, olfactory processing plays a similar role in both male-male and male-female courtship behaviours (electronic supplementary material, figure S6). We monitored the intense-light effect on male-female courtship in sbl and orco mutant flies. Consistent with male-male courtship, sbl male-female courtship behaviour was further increased by intense light, while a less obvious effect on orco was observed because their chasing and wing extension scores were already near saturation at the low light intensity.

Spatio-temporal properties of male-male courtship behaviours
In addition to courtship event statistics shown in figure 2, we also monitored continuous activity patterns among the male flies, using IowaFLI Tracker [40] for an automated analysis of kinematic properties as well as social interactions among individually identified flies. As figure 3a shows, WT flies, upon high-intensity illumination, displayed a drastic increase in activity levels (panel (i), locomotion tracks, with individual flies colour-coded) as well as a greatly enhanced level of royalsocietypublishing.org/journal/rsob Open Biol. 13: 220233 social interactions ( panels (ii), interactograms, with coloured time segments registering other individuals active within the 3.75 mm vicinity; an interaction proximity criterion of 3.75 mm between centroids was adopted because it optimally captures chasing events in WT flies). By contrast, norpA flies, upon high-intensity illumination, displayed neither a drastic increase in activity levels ( panel (i)) nor a greatly enhanced level of social interactions ( panel (ii)). No male-male chasing or wing extension events were evoked by intense light (figure 2d). Interestingly, orco flies exhibited a contrasting case for reduced response to intense light. Chasing or chaining events already occurred at low light intensities, indistinguishable from behavioural activities under intense light, as indicated by the locomotion tracks ( panel (i)) and interactograms ( panel (ii)).
In figure 3, a selected area (boxed) in the interactogram panel for each genotype is enlarged to illustrate interaction records for individually identified flies ( panel (iii)). Each fly in the arena was assigned a number and a colour code. In each colour-shaded strip for an identified fly, the other flies in the vicinity of this individual are recorded as bar segments of darker colours. Thus, the reciprocal relationship among the colour-coded individuals can be traced for each time point in the original video record (15 frames s −1 ). The interactogram provides the raw source data for time-stamped proximity information for the involved individuals. Following visual inspection, particular categories of courtship interactions (wing extension, chasing, and chaining) could be related back to the video recording and the locomotion tracks to obtain a more comprehensive description of the event.
IowaFLI Tracker also provides statistics for kinematics parameters, such as distance travelled, speed, and % time moving (electronic supplementary material, table S1). From the table, norpA is clearly hyperactive (with high values of speed, distance travelled, and % time moving). However, most of the recorded norpA interactions, defined by the 3.75-mm proximity criterion, were not courtship-related and occurred most often along the arena periphery (figure 3b(i)). By contrast, many interactions indicated in the interactograms for WT and orco could be related to compact bundles of locomotion tracks traversing across the arena for the same time segments (figure 3a(i)(ii),c(i)(ii)). Our findings add a striking case of male-male courtship behaviour in WT flies, in contrast to previous reports from a number of genetic variants, including fru [25,41], painless [35], an unidentified mutation (on Chr. 3L) reported by Sharma [52], as well as several mini-w + transformants [38,39]. A strong visual contribution to such behaviours was suggested by Sharma's observation that blue light inhibits male-male courtship [52]. Similarly, blue and UV lights have been reported to suppress male-female courtship [6]. In a preliminary follow-on experiment, we subjected orco 1 and orco 2 males to intense blue light and found that male-male courtship was severely suppressed (electronic supplementary material, table S2). It is important to note that the two orco alleles employed in this study carry the mini-w + construct (in place of the orco gene) in a w − background (see Methods for the construct). Their eye colour appeared normal, indicating strong mini-w + expression. Therefore, the extreme phenotypes of w;;orco 1 and w;;orco 2 in this report may have had contributions from mini-w + expression. Our preliminary analysis of orco 1 and orco 2 heterozygotes (orco/+) generated from crossing w/Y; orco males to w 1118 females displayed a correlation between eye-colour density and male-male courtship activity. We observed darker eye colour in w;;orco 1 /+ than w;;orco 2 /+, indicating differences in mini-w + expression, and accordingly a stronger male-male courtship behaviours in w;;orco 1 /+ (electronic supplementary material, table S3). In terms of male-male courtship scores, we observed in the following sequence as shown in electronic supplementary material, table S3: w; orco 1 =orco 1 ≃ w; orco 2 =orco 2 . w; orco 1 =þ . w; orco 2 = þ : Furthermore, norpA P12 /Y;;orco 1 /+ and sev LY3 /Y;;orco 1 /+ males have been observed to behave like norpA P12 and sev LY3 males, suggesting total suppression of the orco 1 /+ phenotype by the visual mutations, (electronic supplementary material, table S3). Taken together, our results confirm a critical role of visual system function in male-male courtship.

The effective light intensity range and underlying candidate neural circuits
The above findings demonstrate that light plays a major role in courtship behaviour in Drosophila melanogaster. Besides facilitating visually guided motor behaviours, intense light serves as a potent modulator promoting male sex drive as indicated by increased courtship behaviour towards males as well as females. Notably, the light intensity range required to effectively trigger male-male courtship (between 2 and 6 klx; electronic supplementary material, figure S3) is several fold greater than common light sources encountered in the laboratory, including room lights or incubator lighting (approx. 0.5 klx, measurements in our laboratory). However, this light intensity is within the range that flies encounter in the wild, as direct sunlight can be above 100 klx [53]. Importantly, our findings with both incandescent light and sunlight (figure 2c) indicate a variety of sufficiently intense broad-band light sources (electronic supplementary material, figure S4) can induce male-male courtship. It is tempting to speculate based on recent advances in identification and manipulation of neuronal components of various circuits involved in male courtship behaviour. Our analysis suggests an interplay of visual and olfactory processing. Indeed, in a model previously proposed by Wang et al. [35], male flies have an intrinsic drive to court that is normally restrained by olfactory processing of certain signals from other males (e.g. cisvaccenyl acetate [22]). Flies deficient in olfactory processing such as orco and sbl would lack these restraints, consistent with the increased male-male courtship shown in figures 2 and 3. Conceivably, intense light could serve to unleash the intrinsic courtship drive either by supercharging the courtship motor programme to overcome olfactory inhibition or by directly suppressing the inhibitory olfactory processing in WT flies.
Within the broad-band illumination used in this study, the shorter wavelength UV and blue light preferentially activate R 7 and R 8 , respectively. UV and blue light are known to suppress male-female courtship [6] and blue light was found to inhibit male-male courtship in the mutant flies reported by Sharma [52]. Our analysis of norpA and sev mutants implies that R 7 and/or R 8 processing plays an important role in lightinduced courtship (figure 2d). Photoreceptors R 7 and R 8 directly target several classes of medulla neurons, including Dm8 amacrine-like cells, and Tm5 and Tm9 cells which project to the lobula plate [54]. It is possible that these medulla neurons directly or indirectly influence excitability of fru-expressing P1 neurons in the lobula plate, which exert strong modulation on the activity of LC10a circuit that has been shown to be essential for controlling courtship chasing performance [3,55]. Future analysis will elucidate the action and modulation of the neural circuit underlying light-evoked male-male courtship. Our findings provide a new context of courtship behaviour and can add constraints for data interpretation in the studies of neural circuits responsible for the repertoires in male courtship behaviours.

Courtship behaviour recording
Fly courtship behaviour was observed in a custom-built chamber which consisted of an arena piece, and arena ceiling royalsocietypublishing.org/journal/rsob Open Biol. 13: 220233 cover, and a light-shielded cylindrical chamber (figure 1a). The arena piece consisted of a Plexiglas sheet (2 mm thick) with four milled circular chambers referred to as 'regions of interest' or ROIs (26 mm in diameter) seated on filter paper (Whatman No. 1) forming 2-mm-high behavioural arenas (4 ROIs). The arena ceiling cover was a Plexiglas sheet of the same specification. The light-shielded chamber was constructed from a 4inch PVC pipe coupler (NIBCO 4801) with a custom milled PVC lid. A 21 LED strip (STN-A30K80-A3A-10B5M-12 V, SuperBrightLEDs.com) was glued along the inner rim next to the base of the light chamber. LEDs were powered by a custom-built current-controlled power supply (electronic supplementary material, figure S7). For figure 2c experiments, incandescent light was delivered via fibre optic light pipes from an USHIO EKZ 10.8 V, 30 W bulb. Spectral characteristics of the above light sources were determined across a range of power supply currents using a flame miniature spectrometer (Ocean Optics, Orlando, FL; electronic supplementary material, figure S4a-c) or Goyalab GoSpectro (Axiom Optics, Somerville, MA; electronic supplementary material, figure S4d). The spectral power across a range of wavelengths (λ = 189 nm to 884 nm) was measured following manufacturer's instructions. In electronic supplementary material, figure S4, spectral data were normalized to the peak emission detected (i.e. E(λ)/ E(peak) for the wavelength λ). In addition, the spectral characteristics of sunlight on a sunny spring day (14.00, 10 April 2023, in Tuscaloosa, AL, USA) were measured (electronic supplementary material, figure S4).
Fly behaviours were captured by a Logitech c920 webcam connected to a PC. For microphone recordings (figure 1b), a modified arena of a larger dimension was constructed with a hole drilled in the side of the arena. A high-gain microphone (FC-23629-P16, Knowles Electronics, Itasca, IL, USA; see also [60]) was placed in the opening to pick up fly sounds. Light intensity measurements (electronic supplementary material, figure S2) were performed using a digital light meter (DLM1, Universal Enterprise Inc., Portland, OR, USA) following manufacturer's instructions.
Behavioural recordings were performed by aspirating 8 flies into each arena (without anaesthesia). Following a brief acclimatization period (less than 5 min), flies were recorded for 2 min under 'low-light' conditions, immediately followed by 2 min of 'high-light' conditions. In some experiments (figure 1d,e), we included a 2-min 'low-light' recovery period.

Quantification and statistical analysis
Behavioural scoring was carried out in groups of 8 flies confined in the arena (for male-female courtship, 4 males and 4 females were used). Criteria for chasing and wing extension behaviours were based on Hall [20], and we defined chaining behaviour as the formation of a mobile chain of 4 or more flies (cf. Zhang & Odenwald [38] and Hing & Carlson [39]). Scoring was performed by a pair of operators (an observer and a recorder), either on-site during live video recording or by viewing playback of videos. Recording and scoring were performed on pairs of ROIs, one for experimental and one for control (or comparison) flies. Each successive 10-second interval in the 2-min observation periods was divided into two 5-second scoring windows and experimental and control ROIs were scored alternately (i.e. 5 s for one ROI followed by 5 s for the other). The presence or absence of each of the courtship behaviours in an ROI was recorded in the 5-second window. The total number of 5-second windows (out of 12 possible) in which the respective behaviours were observed were reported as the 'total score', ranging from 0 to 12. In addition, automated analysis of walking kinematics was performed offline with IowaFLI Tracker as described in Iyengar et al. [40].
All statistical analyses were performed in MATLAB r2021b. Unless otherwise indicated, all statistical significance values are reported from paired t-tests comparing the 'low' versus 'high' light conditions. For comparisons across multiple groups (figure 1e; electronic supplementary material, figure S2d), a one-way ANOVA was performed as a preliminary step. Electronic supplementary material, table S4, indicates the complete list of statistical analyses and results in this study.
Data accessibility. An updated version of IowaFLI Tracker (version 3.1) and the original data sets for figure and table construction can be found on github (https://github.com/IyengarAtulya/IowaFLI_tracker).
Additional information is provided in electronic supplementary material [61].